EFFECT OF ENZYMATIC MACERATION ON SYNTHESIS OF HIGHER ALCOHOLS DURING MANGO WINE FERMENTATION L.V.A. REDDY 1,2 and O.V.S. REDDY 2,3 1 Department of Microbiology Yogi Vemana University Kadapa 516003, India 2 Department of Biochemistry Sri Venkateswara University Tirupati 517 502, India Received for Publication April 29, 2007 Accepted for Publication February 27, 2008 ABSTRACT Mango is a popular and highly produced fruit in India. Wine from two mango cultivars Banginapalli and Totapuri was prepared and the mode of higher alcohols synthesis during wine fermentation was evaluated. Mangoes were peeled off and juice was extracted immediately after crushing (control) and also after 10 h of pectinase treatment. The recovered juice was allowed to ferment at 15 and 20C. During fermentation, higher alcohols and sugars were measured. Contents of all the higher alcohols analyzed increased during fermentation. More volatiles were observed in wine produced from Totapuri cultivar (358 12.7 mg/l) than wine from Banginapalli cultivar (340 10.5 mg/l). Pectinase treatment increased the synthesis of iso-amylalcohol, 2-phenyl ethanol, n-propanol, n-butanol and methanol during fermentation. Sensory evaluation scores of wine correlated to the sum of higher alcohols. The results of the present study suggest that the pectinase treatment increases the mango juice yield and results in better wine sensory quality. PRACTICAL APPLICATIONS Nowadays, the number of wine consumers is increasing rapidly all over the world. In order to meet the demand, researchers are developing alternative wine producing technologies. In the present study, we made an attempt to 3 Corresponding author. O.V.S. Reddy, Department of Biochemistry, Sri Venkateswara University, Tirupati-517-502, A.P., India. TEL: +91-9440046293; EMAIL: lvereddy@yahoo.com 34 Journal of Food Quality 32 (2009) 34 47. 2009 Wiley Periodicals, Inc.
HIGHER ALCOHOL SYNTHESIS DURING MANGO WINE PRODUCTION 35 produce wine from mango, the king fruit of world. The main objectives of this study are to increase the quality and quantity of mango juice for wine production and evaluate the aroma compounds kinetic parameters during wine fermentation. This study also provides an adequate process for both juice and wine production from mango and other tropical fruits. The findings regarding pectinase treatment, formation of major volatile compounds and sensory evaluation can be valuable references to the wine industry. INTRODUCTION Wine production and consumption is quite popular all over the world and also one of the ancient practices. Although grapes are the main raw material used for the wine production, there is an increasing interest in the search of other fruits, such as apricot, apple and palm sap, suitable for wine making. In countries where grapes are not abundantly available, they are alternatively producing the wine from their local fruits that are cheap and readily available (Onkarayya 1986; Reddy 2005). India is the largest producer of mango in the world with 59% share in the total world production. Mango production has experienced continuous growth in the last decades of the 20th century (Onkarayya 1986; Baisya 2004). Production of wine from mango is one of the alternative ways to use and convert surplus production into a valuable product (Onkarayya 1986; Reddy 2005). Wine production involves several stages. During the alcoholic fermentation stage, wine yeasts, belonging mainly to Saccharomyces cerevisiae species, catalyze the conversion of sugars (from fruit juice) into ethanol, carbon dioxide and minor metabolites that will give flavor to the final product. Flavor is a combination of taste and aroma, and is of particular importance in determining food preferences. Many factors influence the flavor of the food products. In the case of wine, the factors that affect flavor and the principal differences between a cheap wine and the most expensive wine are of obvious interest (Bauer and Pretorius 2000). Volatile compounds in the wine are responsible for aroma because they have greater vapor pressure. These compounds include several higher alcohols and their esters that are synthesized during fruit ripening, juice fermentation or later, during storage of the wine in wooden casks or in bottles. Among all flavor compounds present in wine, higher alcohols occupies first place on quantity basis and also influence certain sensory characteristics of wine. Higher alcohols are synthesized during fermentation from oxo-acids that are derived as by-products from amino acid and glucose metabolism (Mangas et al. 1994). Ribereau-Gayon et al. (1975) have reported that 10% of total higher alcohols are synthesized from corresponding amino acids, 65% from other amino acids and 25% from sugars. According to
36 L.V.A. REDDY and O.V.S. REDDY Reazin et al. (1973), yeast, Saccharomyces cerevisiae generates amyl alcohol from the amino acid isoleucine and amyl alcohol, iso-amyl alcohols and n-propanol from threonine. Many research groups have investigated the higher alcohol concentration in wine. Jepsen (1978) reported a positive correlation with n-butanol to the scores of aroma quality of apple juice. Some higher alcohols, particularly iso-amyl acohol and iso-butanol, have been considered to be the most important flavoring agents in the wine (Rous et al. 1983). Rapp and Mandery (1987) found the concentration of total higher alcohols in wine to be in the range 80 540 mg/l and also reported that a concentration of up to 300 mg/l contributes to pleasant flavor, while concentrations above 450 mg/l provoke unpleasant flavor and harsh taste. The aroma compound production during fermentation mainly depends on yeast strain and the temperature (Garcia et al. 1999; Castellari et al. 1995). Traditional wine manufacturing uses wooden mills to crush the fruit and batch mechanical pressers to extract the juice and fermentation is carried out in wooden casks without any temperature control. A new approach in wine production involves the use of selected yeast strains, as well as controlled temperature during fermentation. A major conversion of sugars during fermentation provides an increased concentration of higher alcohols in apple cider (Mangas et al. 1994). Fruit maturity also influences the synthesis of higher alcohols during fermentation; higher alcohols concentration was low in Semillon wines made from late harvested grapes (Ribereau- Gayon and Sudarud 1991). Both the spontaneous and enzymatic clarification of apple and grape juice increases the higher alcohol concentration (Mangas et al. 1994; Aragon et al. 1998). Cider taste depends mainly on cultivar (Beech and Carr 1977), while cider odor depends more on the processing technology (Gomis et al. 1991) and yeast strain employed (Leguerinel et al. 1989). There are reports on mango wine production but no reports on higher alcohol concentration and sensory quality. Taking into account the absence of previous studies that tackle the volatile composition and the enological variables of the mango wine, the present paper has been carried out with the aim to produce the quality mango must, to evaluate the higher alcohol synthesis in the wine production from two different mango cultivars with and without pectinase treatment and their influence on sensory properties of wine. MATERIALS AND METHODS Mango Fruits Two varieties of mangoes, Banginapalli and Totapuri were selected, which are abundantly available and grown especially in the southern part of
HIGHER ALCOHOL SYNTHESIS DURING MANGO WINE PRODUCTION 37 India. The selected fruits were procured from local market of Tirupati, Andhra Pradesh, India. Mango Processing Mangoes were washed with 1% HCl and peeled off manually. Pulp was recovered manually and up to 50 mg/l potassium metabisulfite was added in accordance with the degree of maturation and hygienic state of the mango fruits. The pulp was divided into two portions. The first portion was left as a control and the subsequent samples of the second portion were treated with previously optimized enzyme concentration and conditions (0.8% and ph 5, Reddy 2005) of Trizyme P50, pectinolytic enzyme procured from Triton Chemicals, India, Mysore. All the above treatments 200 ml of pulp in 500 ml conical flask were placed on a rotary shaker for intimate mixing for 10 h at 37C. Juice extraction was made by pressing the treated pulp in cheesecloth. The juice obtained in this manner was then subjected to analysis of total and reducing sugars, total acidity, ph and soluble solids content. None of the varieties was ameliorated with sucrose. Microorganism and Inoculum Preparation The ethanol producing yeast, Saccharomyces cerevisiae 101 was obtained from Central Food Technology and Research Institute (India) was used in the experiments. The culture was maintained on MPYD (malt extract 0.3%, peptone 0.5%, yeast extract 0.3%, dextrose 2% and agar 1.5%) slants at 4C. The inoculum was prepared by inoculating the slant culture into 25 ml of the sterile MPYD liquid medium taken in 100 ml Erlen Mayer flask and allowed to grow it on a rotary shaker (100 rpm) for 48 h at 37C. This inoculum (3 10 6 cells/ml) was transferred to 250 ml conical flasks having 100 ml mango juice. Fermentation Batch fermentation of the inoculated must was carried out in a number of flasks by incubating at ph 4.5 and at temperature 20C for 15 days. The samples were collected by separation of the cells by centrifugation at 5,000 g for 10 min. The fermented samples were kept at -20C for a few weeks for chemical and sensory analyses. Analytical Methods The concentration of reducing sugars was estimated by Shaffer and Somogyi (1995) method. Total dissolved solids were measured by estimating specific gravity of water-soluble portion of the juice obtained by the centrifu-
38 L.V.A. REDDY and O.V.S. REDDY gation at 10,000 g for 15 min. The specific gravity was determined at 20C with densitometer. With the aid of approximate tables, the results were converted to grams of dissolved solids per 100 ml and expressed as grams of sucrose. All the values in the present study were mean values of triplicate experiments. Ethanol and other Volatile Compounds Ethanol and other metabolites (glycerol, methanol and total esters) were determined with the help of gas chromatography (Antony 1984). The fermented samples were centrifuged at 5,000 g for 10 min. The supernatant was used for ethanol analysis. An Agilent Systems Gas Chromatograph with Flame Ionization Detector (GC-FID) Model 6890 plus instrument was used and conditions were as follows: 5% Carbowax 20 M glass column on Carbopack-B 80/120 mesh; 6 ft (2 m), 2 mm inner diameter (ID), 1/4 mm nitrogen was used as a carrier gas with a flow of 20 ml/min and the eluted compounds were detected by flame ionization detector (FID); for this the fuel gas was hydrogen with a flow rate of 40 ml/min and the oxidant was air with a flow rate of 40 ml/min; and n-propanol was used as internal standard. Total acidity was determined by neutralization with 0.1 N NaOH expressed in tartaric acid and volatile acidity within the distillate samples expressed in acetic acid mg/100 ml. Sensory Evaluation Test Sensory evaluation of wine samples was performed by six well-trained panelists. The system used was Bux-Baum contains total 20 points including, two points for color, two points for clarity, four points for smell and 12 points for taste (Meilgaard et al. 1999). Statistical Analysis All the experiments were carried out three times (triplicate) and the mean value with standard deviation and significant (P). SPSS, version 11.0 was used for analysis of variance. RESULTS AND DISCUSSION The content of the reducing sugars in the mango juice varied with the cultivar; Banginapalli (18% w/v) mango juice contained more reducing sugars when compared with Totapuri (15% w/v). Pectinase treatment slightly increased the total soluble solids content in both mango cultivars (Table 1). Decreased glucose and fructose levels were monitored, but it was observed
HIGHER ALCOHOL SYNTHESIS DURING MANGO WINE PRODUCTION 39 TABLE 1. EFFECT OF PECTINASE ENZYME TREATMENT ON MANGO JUICE RECOVERY AND COMPOSITION Character Banginapalli Totapuri Untreated Treated Untreated Treated Juice yield (ml/kg) 458 12 550 10 TSS (% w/v) 15 0.60 20.5 0.79 P < 0.001 Reducing sugars (% w/v) 15.8 1.5 18.5 1.6 P < 0.0130 Acidity (% w/v) 0.43 0.06 0.56 0.08 P = 0.0097 ph 4.0 0.53 3.7 0.3 P = 0.2553 416 8.0 500 22 12 0.80 16.5 1.2 P < 0.001 15 1.0 16 1.3 P < 0.1662 0.31 0.03 0.44 0.05 P < 0.0003 4.2 0.5 4.0 0.45 P < 0.4831 TSS, total soluble solids. that in all fermentations, glucose decreased faster than fructose. These results indicate that the yeast that was used for the fermentation in the present study is glucophilic (data not presented). All the pectinase treated juice fermentations were completed in 10 days, while untreated ones needed more than 12 days. It was observed that the pectinase enzyme treatment increased the acidity of the juice. This confirmed the release of galacturonic acid in reaction of pectin methyl esterase on pectin substances. The chemical characteristics of pectinase treated and untreated wines were showed in Table 2. The analyzed higher alcohols have their origin in fruit, except ethyl acetate, iso-amyl alcohol and 2-phenyl ethanol that were metabolized predominantly during fermentation as a result of yeast activity. It was observed that the level of isobutanol increased gradually during fermentation (Banginapalli 58 mg/l and Totapuri 78 mg/l; Fig. 1). More iso-butanol was present in wine produced from cultivar Totapuri, which contains a lower protein level. This observation agrees with the previous reports (Beech and Carr 1977; Ough and Bell 1980). Brown and Ough (1981) reported that the increased isobutanol concentration has a positive effect on the quality of red wines. Pectinase treatment could increase the iso-butanol synthesis by the oxygen diffusion in mash. Mauricio et al. (1997) found an increase of iso-butanol during must fermentation under semi-aerobic conditions in contrast with anaerobic conditions. It is also known that removal of insoluble solids from juice before fermentation significantly reduces the amount of isobutanol (Klingshirn et al. 1987). Kosmerl and Kordis-Krapez (1997) observed an increase in iso-butanol synthesis with higher temperature during must fermentation.
40 L.V.A. REDDY and O.V.S. REDDY TABLE 2. EFFECT OF PECTINASE TREATMENT ON MANGO WINE COMPOSITION Character Banginapalli Totapuri Untreated Treated Untreated Treated Ethanol (% w/v) 6.3 1.1 8.5 1.4 P < 0.0127 Higher alcohols (mg/l) 265 8.2 340 10.5 Total esters (mg/l) 20 1.3 32 1.5 Residual sugars (g/l) 10 1.6 2.5 1 Acidity (% w/v) 0.45 0.04 0.66 0.08 P < 0.0002 ph 4.4 0.50 4.7 0.41 P = 0.2823 5.1 0.9 7.0 1.2 P < 0.0112 279 6.8 358 12.7 16 1.2 25 0.8 15 1.0 3 1.3 0.36 0.03 0.54 0.05 4.5 0.5 4.2 0.5 P = 0.3232 100 Iso-butanol (mg/l) 80 60 40 20 0 0 4 8 12 16 20 Time (days) FIG. 1. SYNTHESIS KINETICS OF ISO-BUTANOL DURING FERMENTATION OF MANGO JUICE FROM BANGINAPALLI AND TOTAPURI CULTIVARS = Banginapalli untreated, = Banginapalli treated, = Totapuri untreated, = Totapuri treated. The most vivid observation in the present study was the 20-fold increase in the content of iso-amyl alcohol in wine compared to mango juice. In the present study, synthesis of iso-amyl alcohol was higher in wine made from Banginapalli (90 mg/l) when compared to Totapuri (79 mg/l) cultivar (Fig. 2). Pectinase treatment enhanced the synthesis of iso-amyl alcohol, because of higher aeration during enzyme treatment. Bosso (1993) found an
HIGHER ALCOHOL SYNTHESIS DURING MANGO WINE PRODUCTION 41 120 Iso-amyl alcohol (mg/l) 100 80 60 40 20 0 0 4 8 12 16 20 Time (days) FIG. 2. SYNTHESIS KINETICS OF ISO-AMYL ALCOHOL DURING FERMENTATION OF MANGO JUICE FROM BANGINAPALLI AND TOTAPURI CULTIVARS = Banginapalli untreated, = Banginapalli treated, = Totapuri untreated, = Totapuri treated. increase in iso-amyl alcohol in white wine obtained from macerated grapes. According to Ohkubo and Ough (1987), synthesis of iso-amyl alcohol depends primarily on fermentation temperature and on juice total nitrogen content. Iso-amyl alcohol below its threshold level contributes a positive effect on wine flavor. Another higher alcohol that is responsible for honey rose flavor and sweet taste of wine is 2-phenyl ethanol, which was increased with the pectinase enzyme treatment (Fig. 3). Wine made from Banginapalli (18 mg/l) contains a higher amount of 2-phenyl ethanol when compared with Totapuri (13 mg/l) cultivar. These results are in accordance with previous reports of Schwab and Schreier (1990) who believed that 2-phenyl ethanol is not derived from the de novo pathway in yeasts, but originates from a glycosidically bound form in the fruits, which is liberated during fermentation. Trace amounts of ethyl acetate were observed in mango juice at the initial stage; it was synthesized de novo and increased significantly during fermentation. Wine made from Banginapalli (20 mg/l) has a higher amount of ethyl acetate compared to Totapuri (13 mg/l) cultivar (Fig. 4). Our results are in agreement with those of Jepsen (1978) who observed synthesis of ethyl acetate in the last phase of beer fermentation. Ethyl acetate is responsible for fruity flavor in wine at very low concentrations. The threshold value for its presence is between 10 12.5 mg/l and higher concentrations above 75 mg/l produces a vinegar smell which negatively affects the wine aroma (Francis and Newton 2005). Clarification of must (juice) prior to the onset of alcoholic fermentation improves the sensory characteristics of white wine. Removal of grape solids
42 L.V.A. REDDY and O.V.S. REDDY 20 2-Phenyl ethanol (mg/l) 16 12 8 4 0 0 4 8 12 16 20 Time (days) FIG. 3. SYNTHESIS KINETICS OF 2-PHENYL ALCOHOL DURING FERMENTATION OF MANGO JUICE FROM BANGINAPALLI AND TOTAPURI CULTIVARS = Banginapalli untreated, = Banginapalli treated, = Totapuri untreated, = Totapuri treated. Ethyl acetate (mg/l) 30 25 20 15 10 5 0 0 4 8 12 16 20 Time (days) FIG. 4. SYNTHESIS KINETICS OF ETHYL ACETATE DURING FERMENTATION OF MANGO JUICE FROM BANGINAPALLI AND TOTAPURI CULTIVARS = Banginapalli untreated, = Banginapalli treated, = Totapuri untreated, = Totapuri treated. from must enhances ester production and limits the release of fusel alcohols (higher alcohols) during alcoholic fermentation, which results in a global increase of wine aroma quality (Moio et al. 2004). n-propanol is not present in all mango juices (Pino et al. 2005). Accumulation of n-propanol was observed at the very beginning of the fermentation
HIGHER ALCOHOL SYNTHESIS DURING MANGO WINE PRODUCTION 43 50 n-propanol (mg/l) 40 30 20 10 0 0 4 8 12 16 20 Time (days) FIG. 5. SYNTHESIS KINETICS OF N-PROPANOL DURING FERMENTATION OF MANGO JUICE FROM BANGINAPALLI AND TOTAPURI CULTIVARS = Banginapalli untreated, = Banginapalli treated, = Totapuri untreated, = Totapuri treated. (Fig. 5). Wine produced from the Totapuri cultivar (43 mg/l) has a high amount of n-propanol compared to wine produced from the Banginapalli cultivar (30 mg/l). Enzyme treatment caused an increase in n-propanol concentration, probably because of abundant O 2 availability (Mauricio et al. 1997). Giudici and Kunkee (1994) found that the n-propanol production strongly depends on yeast strain; S. cerevisiae strain 6527 always produced lower amounts of n-propanol irrespective of the total nitrogen in synthetic medium, while at the same time, strain 6392 responded strongly to the amount of nitrogen in the medium. Methanol is present in all fresh fruits as a result of its liberation from pectin by means of pectin methyl esterase. In the present study, methanol level in the control wine (Banginapalli 80 mg/l and Totapuri 93 mg/l) and the pectinase enzyme treated (Banginapalli 120 mg/l and Totapuri 143 mg/l) process showed a significant statistical difference at the end of fermentation (Fig. 6). A lower amount of methanol was found in wine made from Banginapalli mangoes than Totapuri. Some other authors have suggested that the addition of pectolytic enzymes induces an increase in methanol levels in different fermented products, such as ciders (Massiot et al. 1994) and wines (Reddy 2005). Methanol that remains in wines, is toxic to humans. The human oral lethal dose is 340 mg/kg body weight. Pectolytic enzymes play an important role in the wine fermentation process due to the fact that they improve the extraction of color and aroma compounds (Brown and Ough 1981; Foarty and Kelly 1983). The sensory evaluation test result shows good correlation with
44 L.V.A. REDDY and O.V.S. REDDY 150 Methanol (mg/l) 100 50 0 0 4 8 12 16 20 Time (days) FIG. 6. SYNTHESIS KINETICS OF METHANOL DURING FERMENTATION OF MANGO JUICE FROM BANGINAPALLI AND TOTAPURI CULTIVARS = Banginapalli untreated, = Banginapalli treated, = Totapuri untreated, = Totapuri treated. TABLE 3. SENSORY EVALUATION TEST SCORES OF TREATED AND UNTREATED MANGO WINE* Color Clarity Smell Taste Sum Banginapalli (Untreated) 1.8 1.7 2.8 6.5 12.8 Banginapalli (Treated) 2 2 3.4 8.6 16.0 P < 0.001 Totapuri (Untreated) 1.6 1.8 2.4 6.1 11.9 Totapuri (Treated) 1.8 1.9 3.0 7.8 14.6 P < 0.005 * Data presented was mean of six panelists (n = 6). the chemical and volatile composition (Table 3). Sensory evaluation experiments results are in accordance with previous reports on wine and cider preparations (Mangas et al. 1994; Aragon et al. 1998). The low P-values () of higher total higher alcohols and esters of treated wines show the significant and positive effect of pectinase treatment on wine sensory quality. CONCLUSIONS By the view of our preliminary results, pectinase treatment increases the fermentation performance, as well as chemical component production, during
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